Biology subjects

Soh, H. T.

Publications and source records attributed to Soh, H. T..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Binding and sequestration of poison frog alkaloids by a plasma globulin

Alkaloids are important bioactive molecules throughout the natural world, and in many animals they serve as a source of chemical defense against predation. Dendrobatid poison frogs bioaccumulate alkaloids from their diet to make themselves toxic or unpalatable to predators. Despite the proposed roles of plasma proteins as mediators of alkaloid trafficking and bioavailability, the responsible proteins have not been identified. We use chemical approaches to show that a ~50 kDa plasma protein is the principal alkaloid binding molecule in blood from poison frogs. Proteomic and biochemical studies establish this plasma protein to be liver-derived alkaloid-binding globulin (ABG) that is a member of the serine-protease inhibitor (serpin) family. In addition to alkaloid binding activity, ABG sequesters and regulates the bioavailability of "free" plasma alkaloids in vitro. Unexpectedly, ABG is not related to saxiphilin, albumin, or other known vitamin carriers, but instead exhibits sequence and structural homology to mammalian hormone carriers and amphibian biliverdin binding proteins. Alkaloid-binding globulin (ABG) represents a new small molecule binding functionality in serpin proteins, a novel mechanism of plasma alkaloid transport in poison frogs, and more broadly points towards serpins acting as tunable scaffolds for small molecule binding and transport across different organisms.

physiology↗

bioRxiv2022

A high-throughput strategy for enhancing aptamer performance across different environmental conditions

Aptamers selected under specific environmental conditions (e.g., pH, ion concentration, temperature) often exhibit greatly reduced affinity when used in other contexts. This can be especially problematic for biomedical applications, in which aptamers are exposed to sample matrices with distinctive chemical properties, such as blood, sweat, or urine. We present a high-throughput screening procedure for adapting existing aptamers for use in samples whose chemical composition differs considerably from the original selection conditions. Building on prior work from our group, we have utilized a modified DNA sequencer to screen more than 106 aptamer single and double mutants for target binding under the desired assay conditions. As an exemplar, we screened mutants of a previously reported glucose aptamer that was originally selected in high-ionic strength buffer and exhibits relatively low affinity under physiological conditions. After a single round of screening, we identified aptamer mutants with [~]4-fold increased affinity under physiological conditions. Interestingly, we found that the impact of single-base substitutions was relatively modest, but observed considerably greater binding improvements among the double mutants, highlighting the importance of cooperative effects between mutations. This approach should be generalizable to other aptamers and environmental conditions for a range of applications.

bioengineering↗

bioRxiv2022

Allosteric Regulation of Aptamer Affinity through Mechano-Chemical Coupling

The capacity to precisely modulate aptamer affinity is important for a wide variety of applications. However, most such engineering strategies entail laborious trial-and-error testing or require prior knowledge of an aptamers structure and ligand-binding domain. We describe here a simple and generalizable strategy for allosteric modulation of aptamer affinity by employing a double-stranded molecular clamp that destabilizes aptamer secondary structure through mechanical tension. We demonstrate the effectiveness of the approach with a thrombin-binding aptamer and show that we can alter its affinity by as much as 65-fold. We also show that this modulation can be rendered reversible by introducing a restriction enzyme cleavage site into the molecular clamp domain and describe a design strategy for achieving even more finely-tuned affinity modulation. This strategy requires no prior knowledge of the aptamers structure and binding mechanism and should thus be generalizable across aptamers.

biochemistry↗

bioRxiv2022

Tailoring electrode surface charge to achieve discrimination and quantification of chemically similar small molecules with electrochemical aptamers

Electrochemical biosensors based on structure-switching aptamers offer many advantages because they can operate directly in complex samples and offer the potential to integrate with miniaturized electronics. Unfortunately, these biosensors often suffer from cross-reactivity problems when measuring a target in samples containing other chemically similar molecules, such as precursors or metabolites. While some progress has been made in selecting highly specific aptamers, the discovery of these reagents remains slow and costly. In this work, we demonstrate a novel strategy to distinguish molecules with miniscule difference in chemical composition (such as a single hydroxyl group) - with cross reactive aptamer probes - by tuning the charge state of the surface on which the aptamer probes are immobilized. As an exemplar, we show that our strategy can distinguish between DOX and many structurally similar analytes, including its primary metabolite doxorubicinol (DOXol). We then demonstrate the ability to accurately quantify mixtures of these two molecules based on their differential response to sensors with different surface-charge properties. We believe this methodology is general and can be extended to a broad range of applications.

bioengineering↗

bioRxiv2022

A modular intramolecular triplex photo-switching motif that enables rapid and reversible control of aptamer binding activity

DNA switches that can change conformation in response to certain wavelengths of light could enable rapid and non-invasive control of chemical processes for a wide range of applications. However, most current photo-responsive DNA switches are limited either by irreversible switching or reversible switching with impractically slow kinetics. Here, we report the design of an intramolecular triplex photoswitch (TPS) design based on single-stranded DNA that undergoes rapid and reversible photoswitching between folded and unfolded states through isomerization of internal azobenzene modifications. After optimizing the performance of our photoswitch design, we used molecular dynamics (MD) simulations to reveal how individual azobenzenes contribute to the stabilization or destabilization of the triplex depending on their photoisomerization state. By coupling our TPS to an existing aptamer, we can reversibly modulate its binding affinity with less than 15 seconds of UV light exposure. We further demonstrate reproducible shifting in affinity over multiple cycles of UV and blue light irradiation without substantial photobleaching. Given that our TPS can introduce switching functionality to aptamers without manipulating the aptamer sequence itself, we believe our design methodology should offer a versatile means for integrating photo-responsive properties into DNA nanostructures.

bioengineering↗

bioRxiv2022

Pre-equilibrium biosensors: A new approach towards rapid and continuous molecular measurements

Almost all biosensors that use ligand-receptor binding operate under equilibrium conditions. However, at low ligand concentrations, the equilibration with the receptor (e.g., antibodies and aptamers) become slow and thus equilibrium-based biosensors are inherently limited in making measurements that are both rapid and sensitive. In this work, we provide a theoretical foundation for a novel method through which biosensors can quantitatively measure ligand concentration before reaching equilibrium. Rather than only measuring receptor binding at a single time-point, the pre-equilibrium approach leverages the receptors kinetic response to instantaneously quantify the changing ligand concentration. Importantly, analyzing the biosensor output in frequency domain, rather than in the time domain, we show the degree to which noise in the biosensor affects the accuracy of the pre-equilibrium approach. Through this analysis, we provide the conditions under which the signal-to-noise ratio of the biosensor can be maximized for a given target concentration range and rate of change. As a model, we apply our theoretical analysis to continuous insulin measurement and show that with a properly selected antibody, the pre-equilibrium approach could make the continuous tracking of physiological insulin fluctuations possible.

bioengineering↗

bioRxiv2021

Improved immunoassay sensitivity and specificity using single-molecule colocalization

Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern molecular detection, but the technique still suffers some notable challenges. One of the biggest problems is discriminating true signal generated by target molecules versus non-specific background arising from the interaction of detection antibodies with the assay substrate or interferents in the sample matrix. Single-Molecule Colocalization Assay (SiMCA) overcomes this problem by employing total internal reflection fluorescence (TIRF) microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized fluorescent signals, we can essentially eliminate the confounding effects of background produced by non-specific binding of detection antibodies. We further employed a normalization strategy to account for the heterogeneous distribution of the capture antibodies, greatly improving the reproducibility of our measurements. In a series of experiments with TNF-, we show that SiMCA can achieve a three-fold lower limit of detection compared to conventional single-color assays using the same antibodies and exhibits consistent performance for assays performed in complex specimens such as chicken serum and human blood. Our results help define the pernicious effects of non-specific background in immunoassays and demonstrate the diagnostic gains that can be achieved by eliminating those effects.

biophysics↗

bioRxiv2021

Directed Evolution of Aptamer Discovery Technologies

ConspectusAlthough antibodies are a powerful tool for molecular biology and clinical diagnostics, there are many emerging applications for which nucleic acid-based aptamers can be advantageous. However, generating high-quality aptamers with sufficient affinity and specificity for biomedical applications is a challenging feat for most research laboratories. In this Account, we describe four techniques developed in our lab to accelerate the discovery of high quality aptamer reagents that can achieve robust binding even for challenging molecular targets. The first method is particle display, in which we convert solution-phase aptamers into aptamer particles that can be screened via fluorescence-activated cell sorting (FACS) to quantitatively isolate individual aptamer particles based on their affinity. This enables the efficient isolation of high-affinity aptamers in fewer selection rounds than conventional methods, thereby minimizing selection biases and reducing the emergence of artifacts in the final aptamer pool. We subsequently developed the multi-parametric particle display (MPPD) method, which employs two-color FACS to isolate aptamer particles based on both affinity and specificity, yielding aptamers that exhibit excellent target binding even in complex matrices like serum. The third method is a click chemistry-based particle display (click-PD) that enables the generation and high-throughput screening of "non-nattural" aptamers with a wide range of base modifications. We have shown that these base-modified aptamers can achieve robust affinity and specificity for targets that have proven challenging or inaccessible with natural nucleotide-based aptamer libraries. Lastly, we describe the non-natural aptamer array (N2A2) platform, in which a modified benchtop sequencing instrument is used to characterize base-modified aptamers in a massively parallel fashion, enabling the efficient identification of molecules with excellent affinity and specificity for their targets. This system first generates aptamer clusters on the flow-cell surface that incorporate alkyne-modified nucleobases, and then performs a click reaction to couple those nucleobases to an azide-modified chemical moiety. This yields a sequence-defined array of tens of millions of base-modified sequences, which can then be characterized in a high-throughput fashion. Collectively, we believe that these advancements are helping to make aptamer technology more accessible, efficient, and robust, thereby enabling the use of these affinity reagents for a wider range of molecular recognition and detection-based applications.

bioengineering↗

bioRxiv2021

A system for multiplexed selection of aptamers with exquisite specificity without counter-selection

Aptamers have proven to be valuable tools for the detection of small molecules due to their remarkable ability to specifically discriminate between structurally similar molecules. Most aptamer selection efforts have relied on counter-selection to eliminate aptamers that exhibit unwanted cross-reactivity to interferents or structurally similar relatives to the target of interest. However, because the affinity and specificity characteristics of an aptamer library are fundamentally unknowable a priori, it is not possible to determine the optimal counter-selection parameters. As a result, counter-selection experiments require trial-and-error approaches that are inherently inefficient and may not result in aptamers with the best combination of affinity and specificity. In this work, we describe a high-throughput screening process for generating high-specificity aptamers to multiple targets in parallel, while also eliminating the need for counter-selection. We employ a platform based on a modified benchtop sequencer to conduct a massively-parallel aptamer screening process that enables the selection of highly-specific aptamers against multiple structurally similar molecules in a single experiment, without any counter-selection. As a demonstration, we have selected aptamers with high affinity and exquisite specificity for three structurally similar kynurenine metabolites that differ by a single hydroxyl group in a single selection experiment. This process can easily be adapted to other small-molecule analytes, and should greatly accelerate the development of aptamer reagents that achieve exquisite specificity for their target analytes. Significance statementAptamers offer the exciting potential to discriminate between structurally similar small molecules. However, generating such highly specific aptamers has been proven challenging using the conventional process of counter-selection. In this work, we describe a high-throughput screening platform that can characterize the specificity of millions of aptamers towards a group of structurally related molecules in a single experiment and generate exquisitely specific aptamers without any counter-selection. As exemplars, we generated aptamers with high affinity and specificity towards three structurally related kynurenine metabolites using our platform. Our platform can be readily adapted to other small molecule targets and should therefore accelerate the development of aptamer reagents with exquisite specificity.

bioengineering↗

bioRxiv2021

Real-time monitoring of drug pharmacokinetics within tumor tissue in live animals

The efficacy and safety of a chemotherapy regimen fundamentally depends on its pharmacokinetics. This is currently measured based on blood samples, but the abnormal vasculature and physiological heterogeneity of the tumor microenvironment can produce radically different drug pharmacokinetics relative to the systemic circulation. We have developed an implantable microelectrode array sensor that can collect such tissue-based pharmacokinetic data by simultaneously measuring intratumoral pharmacokinetics from multiple sites. We employ gold nanoporous microelectrodes that maintain robust sensor performance even after repeated tissue implantation and extended exposure to the tumor microenvironment. We demonstrate continuous in vivo monitoring of concentrations of the chemotherapy drug doxorubicin at multiple tumor sites in a rodent model, and demonstrate clear differences in pharmacokinetics relative to the circulation that could meaningfully affect drug efficacy and safety. This platform could prove valuable for preclinical in vivo characterization of cancer therapeutics, and may offer a foundation for future clinical applications.

bioengineering↗

bioRxiv2021

A Metric for Quantifying the Resolution of Molecular Assays

Many assay developers focus on limit of detection (LOD) as a primary performance metric, and LOD is indeed useful for assays designed to determine the presence or absence of an analyte. However, LOD is less useful for continuous assays designed to discriminate between concentrations of an analyte--e.g., glucose monitoring in diabetes, where clinical care is guided by particular concentration ranges and thresholds. In such scenarios, it would be valuable to quantify discriminatory resolution--i.e., whether an assay can differentiate 1 pM from 10 pM-- but no such standardized metric currently exists. Here, we propose a useful solution, termed resolution of molecular concentration (RMC). RMC offers a simple means for characterizing quantitative resolution and quickly comparing the quantitative performance of assays. By raising awareness of the limitations of current metrics for evaluating assay performance, we hope to empower the molecular diagnostics community to evaluate their methods in a more application-appropriate manner.

bioengineering↗

bioRxiv2021

Accelerated electron transfer in nanostructured electrodes improves the sensitivity of electrochemical biosensors

Electrochemical biosensors hold the exciting potential to integrate molecular detection with signal processing and wireless communication in a miniaturized, low-cost system. However, as electrochemical biosensors are miniaturized to the micron scale, their detection sensitivity degrades precipitously, thereby greatly reducing their utility in the context of molecular diagnostic applications. Studies have reported that nanostructured electrodes can greatly improve electrochemical biosensor sensitivity, but the underlying mechanism remains poorly understood, thus making it difficult to fully exploit this phenomenon to improve biosensor performance. In this work, we propose and experimentally validate a novel mechanism in which electron transfer is physically accelerated within nanostructured electrodes due to reduced charge screening, resulting in enhanced sensitivity. We show that this mechanism can be exploited to achieve up to 24-fold increase in signal and nearly four-fold lower limit-of-detection relative conventional planar electrodes. This accelerated electron transfer mechanism should prove broadly applicable for improving the performance of electrochemical biosensors.

bioengineering↗

bioRxiv2021

Discovery of indole-modified aptamers for highly specific recognition of protein glycoforms

Glycosylation is one of the most abundant forms of post-translational modification, and can have a profound impact on a wide range of biological processes and diseases. Unfortunately, efforts to characterize such modifications in the context of basic and clinical research are severely hampered by the lack of affinity reagents that can differentiate protein glycoforms. This lack of reagents is largely due to the challenges associated with generating affinity reagents that can bind to particular glycan epitopes with robust affinity and specificity. In this work, we use a fluorescence-activated cell sorting (FACS)-based approach to generate and screen aptamers with indole-modified bases in an effort to isolate reagents that can differentiate between protein glycoforms. Using this approach, we were able to select multiple aptamers that exhibit strong selectivity for specific glycoforms of two different proteins, with the capacity to discriminate between molecules with identical tertiary structures that differ only in terms of their glycan modifications.

bioengineering↗

bioRxiv2020

Combinatorial Polyacrylamide Hydrogels for Preventing Biofouling on Implantable Biosensors

Biofouling on the surface of implanted medical devices severely hinders device functionality and drastically shortens device lifetime. Poly(ethylene glycol) and zwitterionic polymers are currently considered "gold standard" device coatings to reduce biofouling. To discover novel anti-biofouling materials, we created a combinatorial library of polyacrylamide-based copolymer hydrogels and screened their ability to prevent fouling from serum and platelet-rich plasma in a high-throughput parallel assay. We found certain non-intuitive copolymer compositions exhibit superior antibiofouling properties over current gold standard materials, and employed machine learning to identify key molecular features underpinning their performance. For validation, we coated the surfaces of electrochemical biosensors with our hydrogels and evaluated their anti-biofouling performance in vitro and in vivo in rodent models. Our copolymer hydrogels preserved device function and enabled continuous measurements of a small-molecule drug in vivo better than gold standard coatings. The novel methodology we describe enables the discovery of anti-biofouling materials that can extend the lifetime of real-time in vivo sensing devices.

bioengineering↗

bioRxiv2020

Automated platform for high-throughput screening of base-modified aptamers for affinity and specificity

Aptamers incorporating chemically modified bases can achieve superior affinity and specificity compared to natural aptamers, but their characterization remains a labor-intensive, low-throughput task. Here we describe the non-natural aptamer array (N2A2) system, in which a minimally modified Illumina MiSeq instrument is used for the high-throughput generation and characterization of large libraries ([~]106) of base-modified DNA aptamer candidates on the basis of both target affinity and specificity. We first demonstrate the capability to screen multiple different base modifications to identify the optimal chemistry for high-affinity target binding. We next use N2A2 to generate aptamers that can maintain excellent specificity even in complex samples, with equally strong target affinity in both buffer and diluted human serum. Given that N2A2 requires only minor mechanical modifications to the MiSeq, we believe N2A2 offers a broadly accessible tool for generating high-quality affinity reagents for diverse applications.

bioengineering↗

bioRxiv2020

Rational Design of Aptamer Switches with Programmable pH Response

Aptamer switches that respond sensitively to local variations in pH could enable the development of molecular devices that greatly enhance diagnostic accuracy and therapeutic efficacy. Previous design strategies relied on inserting a known pH-sensitive DNA motif into the aptamer structure. Unfortunately, their performance was ultimately governed by the intrinsic pH-response of the inserted motif and could not be readily tuned to operate across arbitrary pH ranges. In this work, we present a general methodology for converting virtually any aptamer into a pH-responsive switch that can be modulated to undergo a strong change in affinity - in acidic, neutral, or alkaline conditions. This was achieved by inserting two orthogonal motifs that can be manipulated in parallel to tune sensitivity to different pH conditions without altering the core sequence of the aptamer itself. Using a single ATP aptamer, we achieve pH-dependent target binding and release under diverse pH conditions, producing as much as 1,000-fold pH-induced change in affinity. Importantly, we also demonstrate the novel capability to predictably engineer aptamer switches that only exhibit strong target affinity within a narrowly defined pH range. Our approach should offer a highly generalizable strategy for integrating pH-responsiveness into molecular devices.

bioengineering↗

bioRxiv2020

Measuring aptamer folding energy using a molecular clamp

Folding energy offers a useful metric for characterizing the stability and function of aptamers. However, experimentally measuring the folding energy is challenging and there is currently no general technique to measure this parameter directly. In this work, we present a simple approach for measuring aptamer folding energy. First, the aptamer is stretched under equilibrium conditions with a double-stranded DNA "molecular clamp" that is coupled to the aptamer ends. We then measure the total internal energy of stressed DNA molecules using time-lapse gel electrophoresis and compare the folding and unfolding behavior of molecular clamp-stressed molecules that incorporate either the aptamer or unstructured random single-stranded DNA in order to derive the aptamer folding energy. Using this approach, we measured a folding energy of 10.40 kJ/mol for the HD22 thrombin aptamer, which is consistent with other predictions and estimates. We also analyzed a simple hairpin structure, generating a folding energy result of 9.05 kJ/mol, consistent with the value predicted by computational models (9.24 kJ/mol). We believe our strategy offers an accessible and generalizable approach for obtaining such measurements with virtually any aptamer.

biophysics↗

bioRxiv2020

Re-evaluating the conventional wisdom about binding assays

Analytical technologies based on binding assays have evolved substantially since their inception nearly 60 years ago, but our conceptual understanding of molecular recognition has not kept pace. Indeed, contemporary technologies such as single-molecule and digital measurements have challenged, or even rendered obsolete, core aspects of the conventional wisdom related to binding assay design. Here, we explore the fundamental principles underlying molecular recognition systems, which we consider in terms of signals generated through concentration-dependent shifts in equilibrium. We challenge certain orthodoxies related to binding-based detection assays, including the primary importance of a low KD and the extent to which this parameter constrains dynamic range and limit of detection. Lastly, we identify key principles for designing binding assays optimally suited for a given detection application.

bioengineering↗